6,7-Dihydroxycoumarin ameliorates crystal-induced necroptosis during crystal nephropathies by inhibiting MLKL phosphorylation

• Published in: Life sciences (1973) 2021-04, Vol.271, p.119193, Article 119193
• Main Authors: Prajapati, Smita, Tomar, Bhawna, Srivastava, Anjali, Narkhede, Yogesh B., Gaikwad, Anil N., Lahiri, Amit, Mulay, Shrikant R.
• Format: Article
• Language: English
• Subjects: Animal models; Animals; Antioxidants - pharmacology; Antioxidants - therapeutic use; Biocompatibility; Calcium; Calcium oxalate; Calcium Oxalate - toxicity; Calculi; Computer applications; Coumarin; Crystallopathies; Crystals; Cytotoxicity; Deposition; Fibrosis; Fluorescence; Fluorescence microscopy; HEK293 Cells; Histology; Humans; Inflammation; Injury analysis; Kidney Calculi - chemically induced; Kidney Calculi - drug therapy; Kidney Calculi - metabolism; Kidney diseases; Male; Mathematical models; Mice; Mice, Inbred C57BL; Mineralization; Mitochondrial permeability transition; Molecular docking; Molecular Docking Simulation - methods; Molecular dynamics; Molecular modelling; Necroptosis; Necroptosis - drug effects; Necroptosis - physiology; Nephrolithiasis; Oxalate; Oxalic acid; Pathogenesis; Phosphorylation; Phosphorylation - drug effects; Phosphorylation - physiology; Protein interaction; Protein Kinases - chemistry; Protein Kinases - metabolism; Protein Structure, Secondary; Proteins; Rats; Rats, Wistar; Screening; Toxicity; Umbelliferones - pharmacology; Umbelliferones - therapeutic use; Variance analysis; Western blotting
• ISSN: 0024-3205; 1879-0631
• DOI: 10.1016/j.lfs.2021.119193

Mineralization of crystalline particles and the formation of renal calculi contribute to the pathogenesis of crystal nephropathies. Several recent studies on the biology of crystal handling implicated intrarenal crystal deposition-induced necroinflammation in their pathogenesis. We hypothesized that 6,7-dihydroxycoumarin (DHC) inhibit intrarenal crystal cytotoxicity and necroinflammation, and ameliorate crystal-induced chronic kidney disease (CKD). An unbiased high content screening coupled with fluorescence microscopy was used to identify compounds that inhibit CaOx crystal cytotoxicity. The ligand-protein interactions were identified using computational models e.g. molecular docking and molecular dynamics simulations. Furthermore, mice and rat models of oxalate-induced CKD were used for in-vivo studies. Renal injury, crystal deposition, and fibrosis were assessed by histology analysis. Western blots were used to quantify the protein expression. Data were expressed as boxplots and analyzed using one way ANOVA. An unbiased high-content screening in-vitro identified 6,7-DHC as a promising candidate. Further, 6,7-DHC protected human and mouse cells from calcium oxalate (CaOx) crystal-induced necroptosis in-vitro as well as mice and rats from oxalate-induced CKD in either preventive or therapeutic manner. Computational modeling demonstrated that 6,7-DHC interact with MLKL, the key protein in the necroptosis machinery, and inhibit its phosphorylation by ATP, which was evident in both in-vitro and in-vivo analyses. Together, our results indicate that 6,7-DHC possesses a novel pharmacological property as a MLKL inhibitor and could serve as a lead molecule for further development of coumarin-based novel MLKL inhibitors. Furthermore, our data identify 6,7-DHC as a novel therapeutic strategy to combat crystal nephropathies.